Carcinoembryonic Antigen(s) in Liver Disease

Vol. 63. No. I Printed in U.S.A.

(iA STttm:NTEitiJI .OGY

\.n pyri )! ht © 197:! hy Th e William s & Wilkin s Co.

LIVER PHYSIOLOGY AND DISEASE

CARCINOEMBRYONIC ANTIGEN(S) IN LIVER DISEASE I. Clinical and morphological studies TERRENCE MooRE, M.D., PHANI DHAR, M .D ., NoRMAN ZAMCHECK, M.D., AMBROSE KEELEY, M.D ., LEONARD GO'ITLIEB, M.D. , AND HERBERT Z . KUPCHIK, PH.D.

Mallory Gastrointestinal Laboratory, Thorndike M emorial Laboratory, Harvard Medical Unit , Boston City Hospital; the Department of Medicine, Harvard Medical School; Departments of Pathology, Boston University School of Medicine, and Tufts University School of Medicine, Boston, Massachusetts

Using a radioimmunoassay for carcinoembryonic antigen(s), positive assays were obtained in 40 of 88 patients with severe alcoholic liver disease but in none of 14 patients with nonalcoholic liver disease. None of these patients had evidence of gastrointestinal malignancy. Lower levels of serum carcinoembryonic antigen(s) usually were seen in patients with alcoholic liver disease than in patients with colonic or pancreatic cancer. The patients with liver disease and carcinoembryonic antigen-positive sera usually had active alcoholic liver disease with decompensated Laennec's cirrhosis and were readily distinguished clinically from patients suspected to have gastrointestinal malignancy. The radioimmunoassay for carcinoembryonic antigens (CEA) detects small amounts of a circulating glycoprotein in the sera of most patients with cancer of the colon 1 or pancreas 2 and of many patients with other malignancies arising in the gastrointestinal tract. 1• 2 Because the liver helps determine the levels and composition of serum glycoproteins, 3 and

especially as the entodermally derived liver is a source of CEA in the fetus, 4 selected patients with liver disease were studied to determine whether severe nonmalignant liver disorders might influence the radioimmunoassay for CEA. In our initial studies, 24 of 46 patients with severe alcoholic liver disease had detectable CEA activity in their sera and accordingly, caution in the interpretation of the CEA assay for gastrointestinal malignancy in patients with alcoholic liver disease was recommended. 2 We have studied further the use and limitations of the serum CEA assay in 102 patients with liver disease (including biopsy findings in 51 patients) to define clinically and morphologically the liver disorder which yields CEA positivity and to assess implications of these findings for the use of the assay in the diagnosis of gastrointestinal cancer. Results of CEA assays on 44 normal control

Received October 18, 1971. Accepted February 8, 1972. Address for reprint requests to: Dr. Norman Zamcheck, Mallory Gastrointestinal Laboratory, Boston City Hospital, Boston, Massachusetts 02118. This work was supported by grants from the National Cancer Institute (CA-04486 and CA-02090) , the National Institute of Arthritis and Metabolic Diseases (TI AM-5320), and the American Cancer Society (CI-19). The authors express their appreciation to Frank Brown, Richard McCabe, and Richard Souci for their technical assistance. 88

July 1972

89

LIVER PHYSIOLOGY AND DISEASE

subjects and on 86 patients with cancer of the colon or pancreas (reported at the Annual Meeting of the American Federation for Clinical Research at Atlantic City, New Jersey, on May 2, 1971) are included to permit comparisons.

Materials and Methods The patients with liver disease were all inpatients at Boston City Hospital. In reviewing the patients' histories and physical examinations, the duration and degree of alcoholism and the presence or absence of jaundice, hepatomegaly, or stigmata of chronic liver disease, including those reflecting portal hypertension, were noted. The 88 patients with alcoholic liver disease had a mean age of 54.2 years and all had a history of many years, usually decades, of heavy alcohol intake. Nearly all had hepatomegaly, two-thirds had ascites, and one-third had asterixis at the time of testing. The histological diagnosis of the type of liver disease was established within 16 days of drawing blood for CEA assay in 51 patients (by percutaneous needle biopsy in 45 cases and at autopsy in 6 cases). The liver tissue was fixed in either 10% buffered formalin or Carnoys fluid and embedded in paraffin. Sections were stained with hematoxylin and eosin and with Mallory's aniline blue. Grading was performed independently by two pathologists without clinical knowledge of the patients or of their CEA findings. The following features were graded as present or absent in each liver specimen: (a) alcoholic hyalin, (b) fat, (c) hydropic degeneration, (d) bile stasis, (e) portal inf1ammatory reaction, (f) plasma cell component of portal inflammatory reaction, (g) acute necrosis, and (h) acute parenchymal inflammation. Hepatic fibrosis was evaluated as (a) absent, (b) present in portal areas but insufficient for the diagnosis of cirrhosis, or (c) sufficient for the diagnosis of cirrhosis. Cirrhosis was further graded as mild, moderate, or advanced depending on the width and density of bands of fibrous tissue. The radioimmunoassay for CEA was performed according to the method described by Thomson et al. 1 and briefly summarized in a previous publication. 2 The reagents used in the assay were generously supplied by the McGill University Medical Clinic of the Montreal General Hospital. Initially, CEA radiolabeled by Drs. Gold and Freedman 3 was used. Subsequently, CEA prepared in Dr. Gold's laboratory was radiolabeled in this laboratory. Assay results were considered positive if both dupli-

cate tubes of patient's serum contained at least 2.5 ng of CEA per mi. 1 The liver function tests reported were determined within 3 days of drawing blood for CEA assay (usually the same day) . All determinations were done by the biochemistry laboratories at Boston City Hospital. The dry weight of seromucoid (total perchloric acid soluble glycoproteins) was determined by weighing the dialyzed and lyophilized perchloric acid extract of 57 consecutive sera from patients with liver disease or with gastrointestinal cancer and from normal control subjects.

Results Clinical studies. Forty-five per cent of 88 patients with alcoholic liver disease had positive CEA assays whereas none of 14 patients with nonalcoholic liver disease had positive CEA assays. In comparison, 88% of 26 patients with cancer of the pancreas and 72% of 60 patients with cancer of the colon, had positive CEA assays. Two hundred twenty CEA assays on 44 healthy normal control subjects between the ages of 20 and 35 years were all nega~, tive (table 1). Only 17 (19%) of the 88 patients with alcoholic liver disease had values above 5 ng compared with 56 (64% of the 86 patients with cancer of the colon or pancreas (fig. 1). However, no single value clearly separated the two groups. •· The patients with liver disease and positive CEA assays included 30 of 68 patients with Laennec's cirrhosis, 4 of 11 patients with acute alcoholic hepatitis TABLE

1. Carcinoembryonic

antigen(s) 232 patients

Patient group

No. of pati ents

assay on

Positi ve %

Liver disease Alcoholic ..... . ' ' .. Nonalcoholic Cancer confirmed" Pancreas ... . . . .. . . . . . Colon Normal control subjects ...

88 14

45 0

26 60 44

88 72

0

Positive ( + ), duplicate tubes of patient serum contain at least 2.5 ng of carcinoembryonic antigen(s) per ml of serum. • Histologically proved at time of assay. a

LIVER PHYSIOLOGY AND DISEASE

90

60,-----~------------------------,

0..

:::>

0

0:: <0

40

I

u w 20 u.

"'

0

10+

~ -A LCO H OLIC LI VER DISEASE I

-CA NCE R OF COLON OR PANCREAS

FIG 1. Serum carcinoembryonic antigen (CEA) levels in patients with alcoholic liver disease or cancer. Serum CEA levels of 88 patients with alcoholic liver disease (hatched bars) are co mpared with serum CEA levels of 86 patients with histologically confirmed carcinoma of the colon or pancreas. The number of patients in each range of CEA levels is expressed as a percentage of the total group. Levels of CEA above 2.5 ng per ml of patient serum are positive.

without cirrhosis, and 6 of 9 patients with alcoholic hepatitis in whom cirrhosis was not ruled out. The 14 patients with nonalcoholic liver disease who had negative CEA assays included 7 with viral hepatitis, 1 with chronic active hepatitis, 4 with postnecrotic cirrhosis, and 2 with primary biliary cirrhosis. In the patients with alcoholic liver disease, liver function studies done on the 40 patients with positive CEA assays were compared with those obtained on the 48 patients with negative CEA assays. All 88 patients had serum glutamic pyruvic transaminase and bilirubin determinations, 85 had alkaline phosphatase determinations, and 83 had albumin and globulin determinations. The CEA-positive patients had higher mean levels of serum bilirubin (7 .3 mg per 100 ml versus 4.9 mg per 100 ml) and globulin (4.0 g per 100 ml versus 3.5 g per 100 ml) , and lower mean levels of serum albumin (2.8 g per 100 ml versus 3.0 g per 100 ml), transaminase (165 versus 190 U-expressed in milliinternational units with upper limit of normal of 40 U) and alkaline phosphatases (145 versus 159 U-expressed in milliinternational units with upper limit of normal of 85 U) than did the CEA-negative

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patients. However, none of these differences was significant using Student's t-test. Because positive CEA assays have been noted in a few patients with uremia, 2 and because of the occasional association of severe liver disease with azotemia, 58 patients who had blood urea nitrogen determinations within a few days of drawing blood for the CEA assay were divided into CEA-positive and CEA-negative groups. Four of 26 patients with positive assays had modest elevations (above 24 mg per 100 ml) of blood urea nitrogen (35, 28, 25, and 25 ng per 100 ml), and 3 of 32 patients who were CEA-negative had elevations of blood urea nitrogen (63, 27, and 25 ng per 100 ml). Thus, azotemia did not explain CEA positivity in our patients with alcoholic liver disease. The first step in the radioimmunoassay for CEA is the extraction of seromucoid from the patient's sera. Liver disease and malignancies may alter the level and composition of seromucoid, 3 the perchloric acid soluble fraction of serum in which CEA and other glycoproteins are found. To determine whether elevated levels of seromucoid in patients with liver disease might cause nonspecific inhibition in the radioimmunoassay for CEA (and thus explain CEA positivity) , serum CEA levels were compared with the weight of seremucoid (perchloric acid soluble glycoproteins) in the sera of 20 normal control subjects, 12 patients with gastrointestinal cancer, and 25 patients with cirrhosis (table 2). The normal control subjects had negative serum CEA activity. Patients with colonic or pancreatic cancer had a 3-fold increase in seromucoid and a mean serum CEA activity of 6.8 ng per ml. Although the patients with cirrhosis also had higher seromucoid levels than normal (by approximately 50%), there was no difference in seromucoid levels between the CEA-positive and CEA-negative groups. Thus, nonspecific inhibition in the radioimmunoassay by increased amounts of seromucoid did not explain CEA positivity in alcoholic liver disease. The possibility that a change in the seromucoid com-

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LIVER PHYSIOLOGY AND DISEASE

July 1972

2. Comparison of carcinoembryonic antigen(s) (CEA) activity with weight of seromucoid

TABLE

D iagnosis

Serornuco id11 mg/ml

Normal (20)• Gastrointestinal cancer (1 2) . Cirrhosis CEA-negative (6) . CEA-positive (19) .

CEA act ivity 0 ng/ml

0.6

±

0 .3

1. 8

±

0 .5

6. 8

1.0 0.9

±

0.2 0 .3

< 2.5 4. 9 ± 2.3

±

< 2 .5 ±

1 .8

a Expressed as mean ± 1 SE. • Numbers in parentheses indicate numb er of patients.

active alcoholic liver disease. Seven patients with CEA activity greater than 5 ng per ml of serum had Laennec's cirrhosis as well. Within the group of 30 patients with active alcoholic liver disease and Laennec's cirrhosis, highest mean CEA levels were observed in patients whose livers contained the greatest amounts of fibrous tissue (fig. 3) but the differences between any two groups was not significant when analyzed by Student's t-test. In the patients with active alcoholic liver disease, individual morphological features observed in liver biopsies were

NON- AL COHO L IC position occurred without altering the ALCOHO LI C LIV ER LI VER DI S E AS E level of seromucoid but still causing inhibDI SEAS E ition in the assay was not ruled out. Activ e Active Ci rr hosis + In none of the patients with liver disease Cir rhos is 10 did the admitting physician raise the question of the diagnosis of carcinoma of 7 .5 the colon or pancreas. Hepatoma was 8 questioned in a few cases. Most of the patients had a work-up of the gastroin- CEA 5 testinal tract, usually for gastrointestinal ng / ml bleeding. More than 90% of the patients - _ o_- 2.5 - -8-had both upper gastrointestinal series §' co and sigmoidoscopy but fewer than 50% ex= = = had esophagogastroscopy or barium enema. No evidence of cancer was obFIG. 2. Serum carcinoe mbryonic antigen (CEA) served. Four patients, 3 with positive and levels in patients with liver disease. Serum CEA 1 with negative CEA assays, required levels of 48 patients with liver disease are grouped portacaval shunts to control upper gastro- accordin g to the final morphological diagnosis from intestinal bleeding. No evidence of cancer liver biopsy (42 cases) or autopsy (6 cases). was seen at operation and CEA levels did 10 not change following shunt surgery. Fur0 0 thermore, 7 patients with positive CEA assays died of complications of their liver 7.5 disease and at autopsy no evidence of 0 occult malignancy was found in any case. 0 Morphological studies. Liver tissue CEA from 51 patients was examined. Eighteen ng / m l 5 0 0 of 41 patients with alcoholic liver disease 8 0 g 0 had positive CEA assays whereas all 7 2 .5 patients with nonalcoholic liver disease - - - ~had negative assays (fig. 2). Three patients 00 ffi 00 with abnormal liver function tests but with no diagnostic abnormality in liver Mi ld Mo der ate For adva nced F IB ROS I S biopsy specimens had negative CEA assays. FIG. 3. Serum carcinoembryonic ant igen (CEA) All of the positive assays occurred in 18 levels co mpared with the amount of fibrosis in the of 37 patients whose liver biopsies showed liver tissue of 30 patients with Laenn ec's cirrhosis. 0 0

0 0

-

- 0-

-

00 00 00

-

00

0

0

0

0

I

---t----t

92

assessed as "present" or "absent" and the mean CEA values in the two groups were compared using Student's t-test. The presence of alcoholic hyalin correlated with CEA positivity (P < 0.05). Seven patients had CEA values greater than 5 ng per ml of serum (shown in fig. 4 as closed circles). Liver biopsies from these patients showed alcoholic hyalin and fat in all cases and hydropic degeneration in 6 of 7 cases. The one tissue which did not show hydropic degeneration was obtained at autopsy. CEA values under 3 ng per ml were found in all of 4 patients without fat and in 7 of 8 patients without hydropic degeneration of hepatocytes. Elevated CEA levels did not correlate with bile stasis, portal inflammatory reaction, plasma cell component of portal inflammatory reaction, acute necrosis, or acute parenchymal inflammation.

I ALCOHOLIC

HYALINj

,.

Absent

Present

10

·:·

7.5

5 2 .5

Cb

8

-- -~;~~;-

___85\:l_o_____

~A] Absent

~ Present

10

=·

::

7 .5

CEA ng/ml

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LIVER PHYSIOLOGY AND DISEASE

5 2 .5

_ _ DQ_ _ _ _ _ _

0 0

IHYDROPIC

DEGENERATION!

Absent 10

7.5

•

Present

••

~

•

5

2 .5

---~---00~ oooooco

_ _ _ (l. _ _ _ _

~0

8J

--~-Oo

___

00000000

FIG. 4. Serum carcinoembryonic antigen (CEA) levels in 41 patients with alcoholic liver disease grouped according to the presence or absence of alcoholic hyalin, fat, and hydropic degeneration in liver tissue. Patients with serum CEA levels greater than 5 ng per ml of serum are shown as closPd circles.

Discussion The clinical usefulness of the CEA assay in the diagnosis of gastrointestinal cancer1· 2 • 5 is not impaired by these findings. The patients in this study were selected because of their severe liver disease and not because of the clinical possibility of cancer. CEA positivity was usually observed in the most severely ill patients. Thus, the patients with liver disease and positive CEA assays, in addition to being alcoholic in every case and showing active liver disease with Laennec's cirrhosis on liver biopsy in most cases, usually presented the clinical picture of long-standing alcoholism with malnutrition, frequently infection, and liver decompensation (hepatic encephalopathy, gross ascites, hepatomegaly, and spider angiomata). They could usually be distinguished on clinical grounds alone from patients suspected to have gastrointestinal malignancy. ·Liver biopsy was helpful in ruling out primary or secondary carcinoma in the liver in occasional cases when malignancy was stated as a cause for liver decompensation. Moreover, while there was considerable overlap, the levels of CEA activity in the sera of patients with liver disease were usually not as high as those observed with gastrointestinal malignancy. Fourteen patients with nonalcoholic liver disease, selected because of either severe inflammation of the liver (8 cases) or established cirrhosis (6 cases), had negative CEA assays. However, too few patients were studied to rule out entirely the possibility that liver disease activity, rather than activity of alcoholic liver disease per se, produced the abnormal levels of antigen. Two plausible explanations for CEA activity in the sera of patients with alcoholic liver disease are: (1) that the damaged liver is less efficient in removing a glycoprotein with CEA activity produced outside the liver so that this glycoprotein accumulates in serum to levels detectable by the CEA radioimmunoassay, or (2) that the liver, damaged by alcoholism, synthesizes abnormal glycoprotein with CEA

July 1972

LIVER PHYSIOLOGY AND DISEASE

activity, perhaps as part of its reparative growth. The liver as the site for CEA degradation. The liver has been shown to screen out antigens which reach it via the portal venous system from the gut. 6 Patients with advanced cirrhosis have been shown to have elevated serum levels of antigenic proteins. 7 The mechanism for this may be either bypass of the liver via portal systemic shunting of blood or a failure of damaged liver to "inactivate" antigen. To explain detectable CEA-like glycoprotein in the sera of patients with alcoholic liver disease it is necessary to postulate that nonmalignant tissues of these patients produce the same glycoproteins in small amounts that gastrointestinal malignancies produce in much larger amounts. Martin and Martin 8 have demonstrated an antigen in nonmalignant colon which cross-reacts with CEA and Burtin 9 has identified circulating antibodies against CEA in a few patients without colonic cancer (see "Addendum"). However, too little is known of the metabolism of CEA at present. Recently, Shuster et al. (Metabolism of carcinoembryonic antigen, in preparation) have shown a rapid disappearance of aggregate-free radio-labeled CEA from the serum of dogs and rabbits with its coincident accumulation in the liver and have suggested that the liver may be important in the metabolism of CEA. The liver as a source of CEA. Most normal serum glycoproteins are synthesized in the liver. The level and composition of serum glycoproteins is frequently altered in cirrhosis. 10 Although the levels of seromucoid were elevated in the patients with alcoholic liver disease, the weight of seromucoid was the same for patients who had positive CEA assays as for the CEA negative group. Nonetheless, the damaged liver cell may produce glycoproteins which have immunological groups in common with CEA. An antigen immunologically identical to CEA has been extracted from the serum and liver of patients with alcoholic liver disease and in lesser amounts from the normal liver and characterized by a

93

variety of immunological techniques (see accompanying paper 11). The source and function of CEA in gastrointestinal malignancies and in the patients' sera has not been defined. Gold and Freedman 4 identified CEA in all malignancies arising in the gastrointestinal tract and in normal fetal gut and pancreas between 2- and 6-months gestation and postulated that these glycoproteins were produced by entodermally derived, incompletely differentiated fetal cells, or by "derepressed, dedifferentiated" cancer cells. CEA has been identified on the surface of colonic cancer cells 12 • 13 but no intracellular localization has yet been observed. It is possible that the liver synthesizes the CEA that is found in the sera and tumors of patients with gastrointestinal malignancy. CEA could then function as a "symbody" 14 by interacting with the tumor cell surface and restricting antigenic expression. However, CEA is found in much higher concentration in colonic cancer than in liver, even cirrhotic liver. 6 • 1 5 Moreover, it persists in colonic cancer cells propagated in tissue culture. 13 Thus, in the patients with gastrointestinal malignancy, the tumor, and not the liver, is the principal source of CEA. However, patients with alcoholic liver disease who have positive CEA assays should not be assumed to have occult gastrointestinal malignancy. The glycoproteins responsible for this positivity may be produced by the liver or accumulate in serum because the damaged liver fails to metabolize them.

Addendum Dr. Burtin and co-workers 16 have recently shown that antibodies detected in the sera of patients with (and without) colonic cancer could be absorbed out by normal tissue proteins. They concluded that they had no evidence that these were specific anti-CEA antibodies. REFERENCES 1. Thomson DMP, Krupey J, Freedman SO, eta!:

The radioimmunoassay of circulating carcino· embryonic antigen of the human digestive system. Proc Nat! Acad Sci 64:161- 167, 1969

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LIVER PHYSIOLOGY AND DISEASE

2. Moore TL, Kupchik HZ, Marcon N, et al: Carcinoembryonic antigen assay in cancer of the colon and pancreas and other digestive tract disorders. Am J Dig Dis 16:1-7, 1971 3. Greenspan EM: Survey of clinical significance of serum mucoprotein level. Arch Intern Med 93 : 863-874, 1954 4. Gold P, Freedman SO: Specific carcinoembryonic antigens of the human digestive system. J Exp Med 122:467-481, 1965 5. LoGerfo P, Krupey J, Hansen HJ: Demonstration of an antigen common to several varieties of neoplasia. N Eng! J Med 285:138-141, 1971 6. Chase MW: Immunologic tolerance to defined antigens and haptens. Introductory remarks. Fed Proc 25:145-147, 1966 7. Boulet P, Mirouze J , Barjon P, et a!: Serum electrophoresis in liver cirrhosis. Apropos of 50 cases. Sem Hop Paris 37:415-420, 1961 8. Martin F, Martin MS: Demonstration of antigens related to colonic cancer in the human digestive system. lnt J Cancer 6:352-353, 1971 9. Burtin P: Reported at an American Cancer Society Workshop on carcinoma of the colon. Houston, June 6-8, 1969

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10. Greenspan EM: Survey of clinical significance of serum mucoprotein level. Arch Intern Med 93: 863- 874, 1954 11. Kupchik HZ, Zamcheck N: Carcinoembryonic antigen(s) in liver disease. II. Isolation from human cirrhotic liver and serum and from normal liver. Gastroenterology 63:95-101, 1972 12. Gold P, Gold M, Freedman SO: Cellular location of carcinoembryonic antigens of the human digestive system. Cancer Res 28:1331-1334, 1968 13. Gold P , Krupey J, Ansari H: Position of the carcinoembryonic antigen of the human digestive system in ultrastructure of tumor cell surface. J Nat! Cancer Inst 45:219- 225, 1970 14. Appfel CA, Peters JH: Tumors and serum glycoproteins. The 'symbodies'. Progr Exp Tumor Res 12:1-54, 1969 15. Moore TL, Kupchik HZ, Dhar P, et a!: Carcinoembryonic antigen (CEA) in liver disease (abstr). Gastroenterology 60:700, 1971 16. Collatz E, von Kleist S, Burtin P: Further investigations of circulating antibodies in colon cancer patients: on the auto-antigenicity of the carcinoembryonic antigen. Int J Cancer 8:298-303, 1971